Cilia (also known as flagella) are hair-like organelles protrude from the surface of most eukaryotic cells and are responsible for cellular motility, fluid flow and sensory perception. A large group of human diseases, known as ciliopathies, are caused by cilia dysfunction. The elongated shape of the cilium is supported by a highly conserved structure called the axoneme. In most motile cilia, the axoneme has a 9+2 architecture in which nine doublet microtubules (DMTs) surround a central pair of singlet microtubules (MTs). Bound periodically along the length of each DMT are a variety of MT-associated proteins and complexes that decorate the external and luminal surfaces with different periodicities (8,16, 24, 48 and 96-nm). These protein complexes are found in coherent register along the entire length of the DMT, and loss of the coherence causes impaired motility. How periodicity is established, maintained, and synchronized, especially over a long distance, has been a long-standing question in the field. Each DMT has a distinctive structure with one complete ring of A- tubule and one incomplete ring of B-tubule. How the unique architecture of the DMT is formed in vivo is still unclear. Furthermore, many axonemal components are asymmetrically distributed in both the longitudinal direction and the radial direction among the 9 DMTs. For example, 3 of the 9 DMTs contain a unique ?beak? structure in the proximal B-tubule lumens. To date, the molecular components and biological functions of the beak are unknown. In this proposal, based on the identification of 33 microtubule inner proteins (MIPs) in our recent work using high-resolution cryo-electron microscopy (cryo-EM), we propose to elucidate the functions of individual MIPs during ciliogenesis using Chlamydomonas mutants. The objective of this application is to use a combination of genetic and structural approaches to investigate the architectural principles governing the assembly of DMTs and axonemes. We will focus on three key aspects of the architectural principles with the following specific aims: (1) We will identify the key proteins responsible for maintaining coherent registry between different periodicities, and investigate their mutual dependence, using Chlamydomonas mutants lacking filamentous MIPs, external coiled-coil proteins, and proteins located at interfaces between different repeat regions. (2) We will investigate the molecular mechanism governing B-tubule formation, and test two hypotheses: (i) proteins located at the MT seam, the unique site within the A-tubule, are essential for B-tubule formation; (ii) MIPs located at the outer junction (OJ) function to promote the B-tubule formation by shielding the inhibitory effects of tubulin C-terminal tails at the OJ. (3): We will identify protein components of the beak using high-resolution cryo-EM. Our preliminary data suggest that two main components are tektin filaments and SAXO proteins. We will investigate their cellular functions and relevance to human diseases using Chlamydomonas mutants and in vitro assays. Most of the proteins studied here have orthologs in human cilia; therefore, our work will provide a molecular basis for understanding the etiology of many human ciliopathies.